Evidence-Based Guidelines for Controlling Ph in Mammalian Live-Cell Culture Systems

ARTICLE https://doi.org/10.1038/s42003-019-0393-7 OPEN Evidence-based guidelines for controlling pH in mammalian live-cell culture systems Johanna Michl1, Kyung Chan Park 1 & Pawel Swietach 1 1234567890():,; A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid–base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the mis- interpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, − including physiological CO2/HCO3 and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems. 1 Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3PT Oxford, UK. Correspondence and requests for materials should be addressed to P.S. (email: [email protected]) COMMUNICATIONS BIOLOGY | (2019) 2:144 | https://doi.org/10.1038/s42003-019-0393-7 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0393-7 iomedical laboratories routinely perform cell culture to Results Bproduce a cellular environment that is precisely defined, Monitoring culture medium pH under incubation. Buffers are well controlled and physiologically relevant. Among the included in culture media to control acidity, yet the ensuing main chemical variables of culture systems is the concentration pH is not routinely monitored. This becomes a quality control of H+ ions, oftentimes referred to as protons. These ions issue whenever the components of buffering are being disturbed: are present in every aqueous compartment, not least from the for example, in response to metabolic acid production, or as ionization of water. Various solutes can become protonated, a consequence of transferring media between atmospheres of + thereby establishing multiple chemical equilibria involving H different CO2 partial pressures (pCO2). The dye Phenol Red ions. Consequently, the concentration of free H+ ions is (PhR) is routinely included in the media to allow investigators not intuitive to predict, but fortuitously simple to measure to assay medium acidity14,15. Such assessment could be done (e.g. with electrodes or indicator dyes). For over a century, the ‘by eye’, but a more quantitative readout of pH is obtainable pH scale has been the reporting standard for the concentration from the PhR absorbance spectrum, which can be recorded of free H+ ions1, that is, the form that is able to protonate on plate-reader platforms with an incubator chamber (e.g. targets and post-translationally modify proteins, such as Cytation 5, BioTek). To obtain a calibration curve, freshly pre- 2–4 enzymes or receptors . The much larger pool of buffered pared standards of known pH were scanned in a CO2-free + fl H ions can, however, in uence pH through dynamic re- atmosphere to prevent the acidifying effect of CO2. Calibration 5 − equilibration . solutions had no added HCO3 , as otherwise this basic sub- There is still a widely held misconception that buffers have stance would have reacted slowly with H+ ions and then escape fi an inherent ability to set the pH of a solution to a pre-de ned as CO2 gas. Fig. 1a shows absorbance spectra of PhR in level. More accurately, in a system of one dominant buffer, bicarbonate-free Dulbecco’s modified Eagle’s medium (DMEM) pH is related to the concentration of the buffer’s protonated (D7777, Sigma-Aldrich) supplemented with 10% foetal bovine (HB) and unprotonated (B) forms, and the acid dissociation serum (FBS) and 1% penicillin–streptomycin (PS; 100 U mL−1 −1 constant (pKa): penicillin, 0.1 mg mL streptomycin), 10 mM HEPES and 10 mM MES (2-(N-morpholino)-ethanesulfonic acid), and titra- ½B ted to a target pH with NaOH. To correct for pH-independent pH ¼ pK þ log : ð1Þ a ½HB variables, such as light path or PhR concentration, absorbance was sampled at two wavelengths that respond differently to Consider the dissolution of HEPES buffer (4-(2-hydroxyethyl)- pH. Good resolving power is attained by rationing absorbance 1-piperazineethanesulfonic acid), typically supplied as a powder at 560 and 430 nm (Fig. 1b). The best-fit equation can then be of the free acid form. This produces an acidic solution that used to convert PhR ratio assayed in subsequent experiments must be titrated (with base, e.g. NaOH) to the desired pH; once to pH. the [B]/[HB] ratio is raised to the required level, pH will − remain stable, unless there is an additional source of acid or Setting medium pH by pCO2 and [HCO3 ]. In principle, it is base. In live-cell culture, pH disturbances are an inescapable possible to control pH with one of many commercially available consequence of metabolism and there is a general tendency for buffers, but the most physiologically relevant one is CO /HCO −. fi 2 3 media to undergo acidi cation, the extent of which is also a Incubators maintain a CO2-rich atmosphere (typically 5%) to function of medium buffering capacity. In a closed system, it can enable CO /HCO − buffering. A salt of HCO − must be included 2 3 3 + be derived mathematically (see Supplementary Note) and in the medium to balance the spontaneous H -yielding CO2 shown empirically5 that peak buffering capacity is attained hydration reaction, and stabilize pH: ’ when the buffer s protonated and unprotonated forms are ðÞ$ ÀðÞþþðÞ: ð Þ equimolar, that is, when medium pH aligns with the buffer’s CO2 gas HCO3 aq H aq 2 pKa. Many exogenous buffers are available, covering a wide pH Conveniently, pH can be titrated in the range between ~6 and = − range, including HEPES (pKa 7.3; 37 °C), PIPES (piperazine-N, ~8 by varying the concentration of HCO . Figure 1c plots the ′ = − 3 N -bis(2-ethanesulphonic acid); pKa 6.7) and MES (2-(N- relationship between pCO2, [HCO3 ] and pH measured in = 6 morpholino)-ethanesulfonic acid; pKa 6.0) . DMEM (D7777, Sigma-Aldrich) containing 10% FBS and 1% A more active means of maintaining high buffering capacity PS. To keep osmolality constant, any reduction in NaHCO3 was involves the regulation of [HB] and [B], so that their ratio matched by a compensatory rise in NaCl. Over the alkaline is kept at an optimum5. This strategy underpins the rea- range, pH reported by PhR is in very close agreement with the − son why complex organisms rely on CO2/HCO3 buffer (despite prediction of the Henderson–Hasselbalch equation (Eq. 1), = 7 − = low pKa 6.15) , and have evolved gas exchange surfaces consistent with CO2/HCO3 being the dominant buffer (pKa −1 7 (e.g. lungs) and ion transport epithelia (e.g. kidneys) to empower 6.15; CO2 solubility 0.024 M atm , i.e. 1.2 mM in 5%) . − 8 − CO2 and HCO3 homeostasis . The combination of CO2 (an However, at low [HCO ], Eq. 1 underestimates pH. This − 3 acidic gas) with HCO3 (a base) produces quantitatively the discrepancy arises because the so-called ‘intrinsic’ buffers in most important buffer in extracellular body fluids. In culture medium (such as proteins included in serum) react with H+ systems, this so-called carbonic buffer is stabilized by adding an ions generated by CO hydration, pushing the equilibrium − 2 − + amount of HCO3 salt to media and enriching the incubator's (Eq 2) towards a higher [HCO ] and lower [H ], that is, a − 3 atmosphere with CO2. Here, we relate HCO3 and CO2 with less acidic medium. This correction is derived mathematically in − pH, show how the system responds to changes in its components the Supplementary Note. Thus, the concentration of HCO3 and demonstrate how the equilibrium is affected by non-volatile required for attaining a target pH is: ÂÃ buffers (NVBs) added to augment buffering capacity. Further- À À : ¼ ½´ pHtarget 6 15 þ β ´ À : : more, we explain how certain cell culture manoeuvres may lead HCO3 CO2 10 intrinsic pHtarget 7 4 fi to poor pH control. While a number of high-pro le guidelines ð Þ relating to cell culture have been published recently9–13, they 3 do not comprehensively cover the aforementioned issues per- By best fitting the data to this equation, intrinsic β −1 taining to pH. Based on our observations, we propose guidelines buffering ( intrinsic) was estimated to be 1.1 mM pH . Alter- β for good practice in controlling pH in culture systems. natively, intrinsic can be measured empirically from the 2 COMMUNICATIONS BIOLOGY | (2019) 2:144 | https://doi.org/10.1038/s42003-019-0393-7 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0393-7 ARTICLE a b 0.7 Medium pH: 10 5.44 0.6 6.60 7.00 0.5 7.45 8.43 1 0.4 0.3 0.1 0.2 PhR absorbance 0.1 560/430 nm PhR ratio 0.0 0.01 350 400 450 500 550 600 650 456789 Wavelength (nm) Medium pH cd8.0 9 – Eq 1 CO /HCO -free media Corrected Eq 1 2 3 7.5 8 +HCl 5% CO2 7.0 7 7.5 +NaOH 7.0 6.5 6 10% CO Medium pH Medium pH 2 6.5 Buffering capacity 6.0 6.0 5 = 1/slope 5.5 = 1.64 (±0.085) mM/pH 03691215 5.5 4 0 5 10 15 20 25 30 35 40 45 012345 [Bicarbonate] (mM) HCl or NaOH added (mM) Fig.

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